A frequently used test system for developing drugs which control the immune system, such as immunosuppressive agents and immunopotentiators, and also for monitoring changes of the immune system, utilizes antibody production by animals as an index. In one method, erythrocyte is used as an antigen for inducing the antibody production. Erythrocytes of horse and sheep have been sold as a reagent since they are available in large quantities. Among them, sheep erythrocyte (sheep red blood cell: SRBC) is most widely used as an antigenic erythrocyte especially because of its easy storage and handling. In "Seikagaku Jiten (Dictionary of Biochemistry)" (second edition; published by Tokyo Kagaku Dojin), SRBC is explained as an independent lemma whereby it is apparent that SRBC has been widely used.
SRBC as an antigen can easily induce antibody production and cellular immunity by its sole administration without mixing with adjuvants (immunopotentiating substances) and, accordingly, it has been widely used, for example, in immunopharmacological and immunotoxicological studies. As a method of measuring anti-SRBC antibody in blood, a classic method utilizing agglutination is used mostly as a practical measuring method. However, the method has disadvantages in that it is discontinuous and is poor in terms of fine quantitativeness. Another disadvantage is the agglutination is confirmed by the naked eye, so there is no objectivity in the method. Accordingly, various attempts have been made for an objective and more quantitative measuring method, i.e. a method of measuring the anti-SRBC antibody by means of enzyme immunoassay (ELISA). However, the methods which utilize enzyme immunoassay up to now still have some problems to be solved, and are not very accurate, easy and convenient.
For example, a method by Mori et al. hit. J. Immuno-pharmac., Vol. 11, 597-606 (1989)! is a method in which SRBC per se is used as a granule antigen and is directly insolubilized on a plate by a special means. However, turbidity is poor and, moreover, the red color of erythrocytes remains in an insolubilized antigen. Therefore, the Mori, et al. method is not suitable for ELISA wherein the reaction product is measured by means of absorbance using a marker enzyme. In addition, there is a problem with the reproducibility of the Mori, et al. method.
There are other methods such as a method by Temple et al. Fundam. Appl. Toxicol., Vol. 21, 412-419 (1993)! and a method by Van Loveren et al. Int. J. Immunopharmac., Vol. 13, 689-695 (1991)! in which membrane protein of SRBC is solubilized under a specific condition and said soluble protein is used as an antigen for insolubilization. In the method of Van Loveren et al, membrane protein is solubilized by treating with potassium chloride of a high concentration. The substance which is used in those methods is thought to be a part of membrane protein which is solubilized. A problem still remains with respect to the difference in antigenicity between such partial membrane protein and the original SRBC. In addition, the solubilizing and separating operations require time and are troublesome. As such, no suitable insolubilized antigen is used in the methods which have been reported up to now and various problems remain for their practical application.
The present invention provides an enzyme immunoassay for anti-erythrocyte antibody which is objective, reproducible, practical, has excellent quantitativeness and may be performed continuously. The reaction product may be measured by means of absorbance using a marker enzyme. The present invention also provides an antigen for use in enzyme immunoassay which is insolubilized on a plate, has excellent transparency and essentially no turbidity. The antigen does not present the problem of a difference in antigenicity. It is shelf stable for extended periods of time when stored in a dark, cool environment.